The overall goal of this proposed research is to understand the molecular basis of membrane-cytoskeletal interactions in vertebrate cells. The extensive plasma membrane formed by microvilli on the apical surface of the intestinal epithelial cell interacts along the length of the microvilli with the actin cytoskeleton through periodically spaced, ATP-sensitive cross bridges composed of a 110,000-dalton protein-calmodulin complex. This complex also has calcium-activated Mg2+ ATPase activity. To define and characterize the molecular basis of the association of the complex with both the membrane and cytoskeleton and to define the function of the ATPase, the ATPase activity and actin and membrane binding of the complex will be studied under well defined conditions by reconstitution with proteins purified from the microvillus, isolated microvilli and microvillar cytoskeletons and liposomes. Biochemical, biophysical, electron microscopic and immunochemical approaches will be used to study both native and reconstituted systems. The possible function of the ATPase in microvillar motility, in regulation of the cytoskeletal and membrane binding of the complex and in calcium ion transport will be determined. The possible association of the complex with the lipid bilayer of the membrane and with other membrane proteins will be studied. In addition, regulation of these events by calcium ion binding to the complex and by phosphorylation of the 110,000-dalton protein will also be characterized. These studies should enhance our understanding of cytoskeletal-membrane interactions and their regulation in intestinal epithelial and other cells.